Methods and compositions for rapid in vitro propagation of Swertia chirata

ABSTRACT

The present disclosure relates to methods and compositions for in vitro cultivation of species of Swertia, e.g.  Swertia chirata  (Ham.). The disclosure provides culture media comprising Murashige and Skoog (MS) basal culture medium, plant hormones preferably selected from the group consisting of benzyladenine (BAP), gibberellic acid (GA 3 ), and auxins, and other additives, e.g. sucrose and agar. Preferably, auxins are selected from the group consisting of indole acetic acid (IAA), indole butyric acid (IBA), and naphthalene acetic acid (NAA). Individual plant hormone concentrations are preferably from about 0.5 mg/L to about 5.0 mg/L. The disclosure provides methods of in vitro cultivation of  Swertia chirata  comprising contacting preferably axillary bud and/or shoot apex explants with an initiation medium comprising a modified MS basal culture medium, BAP, IAA, IBA, and NAA to produce a primary explant, contacting the primary explant with a shoot propagation medium comprising, a modified MS basal culture medium, BAP, GA 3 , and IAA to produce a secondary explant, contacting a secondary explant with a rooting medium comprising a modified MS basal culture medium, IAA, IBA, and NAA. The methods and compositions of the invention are capable of inducing extraordinarily rapid in vitro propagation of  Swertia chirata . The methods and compositions of the disclosure may be useful for conservation of this threatened species as well as producing bulk quantities, e.g. gram, kilogram or more, of plant material for medicinal applications.

FIELD OF THE INVENTION

The present invention relates to novel culture media compositions forrapid in vitro propagation of Swertia chirata, a threatened plantspecies. Culture media of the invention comprise a modified Murashigeand Skoog (MS) basal culture medium and various plant hormones. Theparticular compositions lead to extraordinarily rapid in vitropropagation of specific parts of Swertia chirata. Mass propagation ofthe plant using compositions of the invention may be achieved by usingaxillary buds and shoot apices in the in vitro culture media. Thepresent invention also relates to methods of rapid in vitro propagationof Swertia chirata.

BACKGROUND OF THE INVENTION

Swertia chirata (Ham) of the Gentianaceae family is a slender, uprightherb found in the Himalayas from Kashmir to Bhutan and Khasi Hills. Theplants is an erect herb, stems are robust 0.6 to 1.5 m, branching leavesare opposite, broadly lanceolate, acute, lower leaf often much larger,sometimes petioled. Calyx and corolla are four-lobed. The corolla isgreen-yellow and tinged with purple. See Kirtikar K M, Basu B D, 1985,Indian Medicinal Plants, Bishen Singh Mahendrapal Singh, Dehradun.

The whole plant contains gentiamine alkaloids and the aerial partcontains xanthones. The main chemical constituents of this plant areophelic acid and chiratin. The plant also contains resins, tannin, gum,carbonates, phosphates and 4 to 6 percent ash. See Kirtikar K M, Basu BD, 1985, Indian Medicinal Plants, Bishen Singh Mahendrapal Singh,Dehradun; Chopra R N, 1982, Indigenous Drugs of India. AcademicPublishers, Calcutta. A number of workers have shown that the drugcontains bitter glycosidal components, chiratin and amarogentin,swerchirin, phytosterol, also a number of acids and phenolic compounds.See Korte F, 1955, Chem. Ber. 88:704; Rorte F, Schicke H G, 1956, Chem.Ber. 89:2404; Dalall S R, Shah R G, 1956, Chemistry and Industry 664.

Swertia species are known for their medicinal value. The Wealth ofIndia, Raw materials Vol X PID (CSIR) New Delhi provide in detail fullaccount of distribution economic importance and uses of genus Swertia.For example, aqueous extracts of Swertia may be used during fever. Otheruses include bronchial asthma, dyspepsia and debility. It is a favouriteremedy in intermittent fevers, acidity and in bilious dyspepsiaaccompanied by fever. Swertia chirata is a valuable medicinal plant.

Due to its high demand by the pharmaceutical industry in India and theworld, the species has been extensively exploited, so much so that it isnow listed a threatened species.

Presently, supply depends on wild sources that have been depleted byover harvesting and progressive habitat clearance. It would, therefore,be helpful to use a tissue culture procedure for large scale propagationand conservation. Beside the strategy evolved should maintain qualityand homogeneity of herbs.

Miura et al (1978) has reviewed the work on in vitro regeneration andthe production of secondary metabolites in Swertia pseudochinensis andSwertia Japonica. These workers have described organogenesis from thecallus cultures of S. japonica and S. pseudochinensis as well asproduction of Swertiamarin in cultured tissues of S. pseudochinensis.

Recently, Keil et al. reported production of Amarogentian in rootcultures of Swertia chirata from untransformed and transformed rootcultures. (Keil et al. 2000 Planta Med, 452-457). Regeneration usingroot explants has recently been described by Wawrosch et al. (1999).

The roots which formed under the regenerated shoots were short andswollen requiring modification of rooting procedure for better survivalrate in the field (Wawrosch C C, Maskay N and Kopp B (1999) Plant CellRep. 18:997-2001). Therefore, new media formulation(s) that are capableof efficient plant regeneration in vitro for mass propagation of Swertiachirata are desirable.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for rapid invitro cultivation of species of Swertia, e.g. Swertia chirata (Ham). Theinvention provides culture media comprising MS basal culture media andplant hormones, preferably selected from the group consisting of indoleacetic acid (IAA), indole butyric acid (IBA), gibberellic acid (GA₃),benzyladenine (BAP), and naphthalene acetic acid (NAA). The inventionprovides methods of Swertia propagation comprising:

-   -   contacting a Swertia explant with a first medium comprising MS        basal culture medium I, from about 0.5 mg/L to about 1.0 mg/L        benzyladenine, from about 0.5 mg/L to about 1.0 mg/L indole        acetic acid, from about 0.5 mg/L to about 1.0 mg/L indole        butyric acid, about 0.5 mg/L naphthalene acetic acid, and from        about 1% to about 3% sucrose to produce a primary explant;    -   contacting this primary explant with a second medium comprising        MS basal culture medium I, from about 0.5 mg/L to about 1.0 mg/L        benzyladenine, about 1.0 mg/L indole acetic acid, about 1.0 mg/L        gibberellic acid, and from about 1% to about 3% sucrose to        produce a secondary explant; and    -   contacting this secondary explant with a third medium comprising        MS basal culture medium I, about 1.0 mg/L indole acetic acid,        about 1.0 mg/L indole butyric acid, about 1.0 mg/L naphthalene        acetic acid, and from about 1% to about 3% sucrose to produce a        tertiary explant.        The methods and compositions of the invention are suitable for        use with axillary buds and shoot apices. The methods and        compositions of the invention are further suitable for mass        propagation of Swertia. The methods and compositions may be used        to achieve consistent plant growth over several generations.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the United States Patent andTrademark Office upon request and payment of the necessary fee.

FIG. 1. Explants obtained from Swertia chirata shoot apices and axillarybuds in response to tissue culture media formulations of the invention.

FIG. 2. Proliferation of multiple shoots on media comprising (A)modified MS basal culture media, 0.5 mg/L BAP and 1.0 mg/L GA₃ or (B)modified MS basal culture media, 1.0 mg/L IAA and 3% sucrose.

FIG. 3. Root induction at the base of in vitro shoot on media comprisingmodified MS basal culture media and IAA.

FIG. 4. Example of a well developed Swertia chirata plantlet propagatedin vitro.

FIG. 5. Plants that were regenerated in vitro according to the inventionafter being transferred to soil comprising a 1:1 mixture of sterilizedgarden soil and vermiculite.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, absent a contrary indication, thefollowing definitions shall apply.

Cultivate—To grow, foster growth or propagate. The term is not limitedwith respect to developmental stage, but encompasses progression fromany developmental stage to any other developmental stage.

Explant—A plant at any development stage, substantially free ofcontaminating microorganisms, suitable for culture in a nutrient medium.

Fast or Rapid—A healthy, robust Swertia chirata plant with a shootlength of from about 1 cm to 5 cm suitable for transplanting to soil maybe produced in vitro from shoot apex or axillary bud explants in about18 weeks or less.

In the wild—Cultivation and/or propagation “in the wild” primarilyrefers to outdoor growth of Swertia plants in an environment similar toSwertia's native habitat substantially free of human manipulation.However, this term encompasses any environment wherein at least one ormore of the following factors is not and/or cannot be controlled:temperature, humidity, photoperiod, light intensity, and exposure tomicroorganisms, herbivores, or pathogenic organisms.

Induce—To stimulate or support a response.

MS basal culture medium—Culture medium described by Murashige T andSkoog F (1965, Physiol Plant 15: 433-497), variants thereof known in theart, and MS basal culture medium I.

MS basal culture medium I—This comprises 1650 mg/L NH₄NO₃, 1900 mg/LKNO₃, 440 mg/L CaCl₂.2H₂O, 370 mg/L MgSO₄.7H₂0, 170 mg/L KH₂PO₄,0.83mg/L KI, 6.2 mg/L H₃BO₃, 16.9 mg/L MnSO₄.4H₂0, 8.6 mg/L ZnSO₄.7H₂0,0.025 mg/L Na₂MoO₄.2H₂0, 0.025 mg/L CuSO₄.5H₂O, 0.025 mg/L CoCl₂.6H₂0,0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl, 0.1 mg/L thiamine, and2 mg/L glycine. Variations of the indicated concentrations, if any, arepreferably less than 5%. The pH is about 5.8.

Time—Period following initial contact with a media. For example, “weeks”may refer to the number of weeks after explants were first contactedwith a medium.

General Remarks

The instant invention relates to in vitro cultivation of species of thegenus Swertia, preferably Swertia chirata, more preferably Swertiachirata (Ham). The present invention is not limited to any particularvariety or genotype but may be applied to genetically diverse varietiesand species of Swertia.

The methods and compositions of the invention may be used to raise largepopulations of genetically heterogeneous Swertia chirata seedlings forreliable and effective in-situ and ex-situ conservation of thisthreatened species. The methods and compositions of the invention mayalso be used to cultivate Swertia chirata anywhere in the world duringany season. Fast in vitro propagation of Swertia chirata according tothe invention may allow wider utilization of its medicinal properties.

The instant invention represents the results of years of Applicants'effort. The concentration ranges of MS culture medium and hormonesprovided by the instant invention have been observed to be of criticalimportance. It is believed that any deviation from the same is unlikelyto produce desired results.

The invention contemplates use of these methods and compositions forcommercial bulk production of Swertia chirata. Bulk production mayresult in gram quantities of this plant and preferably kilogramquantities or more.

Materials

The invention contemplates the use of any plant cells, tissues or organsto initiate in vitro cultures. In some preferred embodiments, axillarybuds and shoots apices are used since these tissues are the mostregenerative and result in multi-fold increase in shoot proliferation.In some preferred embodiments, axillary shoot proliferation may beachieved using shoot apex and axillary shoot bud explants.

The invention provides a synergistic tissue culture media formulation toregenerate Swertia chirata shoot apices and axillary shoot bud explantsin vitro. The formulation was found to be useful for regeneration oflarge number of tissue cultured plantlets of Swertia chirata in vitrovia axillary shoot proliferation utilizing shoot apices and axillaryshoot buds explants in vitro.

The culture media compositions of the invention are preferablyhomogeneous and may be used for culture initiation, shoot propagation,and root formation to produce a large number of Swertia chirata plantsin vitro. The culture media compositions of the invention compriseMurashige and Skoog (MS) basal culture medium, varied concentrations ofplant hormones, and other additives. Plant hormones may be selected fromthe group consisting of IAA, IBA, GA₃, BAP, and NAA. Additives include,for example, agar, sucrose, antibiotics, and antifungal agents. Inpreferred embodiments, the range of plant hormones is from about 0.5mg/L to about 1.0 mg/L for BAP, from about 0.1 mg/L to about 1.0 mg/Lfor GA₃, from about 0.5 mg/L to about 1.0 mg/L for IAA, from about 0.5mg/L to about 1.0 mg/L for IBA, and from about 0.5 mg/L to about 5.0mg/L for α-NAA.

In some embodiments of the invention, a first or “initiation” mediumcomprises MS basal culture medium, from about 0.5 mg/L to about 1.0 mg/LBAP, optionally about 0.5 mg/L NAA, from about 0.5 mg/L to about 1.0mg/L IAA, from about 0.5 mg/L to about 1.0 mg/L IBA, and from about 1%to about 3% sucrose. These compositions are capable of inducing orsupporting initiation of an axillary bud and/or shoot apex explantculture. In some embodiments of the invention, the initiation mediumcomprises modified MS basal culture medium, about 0.5 mg/L BAP, andabout 0.5 mg/L NAA. In some embodiments of the invention, the initiationmedium comprises modified MS basal culture medium, about 1.0 mg/L BAP,and about 1.0 mg/L IAA.

In some embodiments of the invention, a second or “shoot proliferation”medium comprises MS basal culture medium, optionally from about 0.5 mg/Lto about 1.0 mg/L BAP, about 1.0 mg/L IAA, about 1.0 mg/L GA₃, and fromabout 1% to about 3% sucrose. These compositions are capable of inducingor supporting a high shoot proliferation and development with shootmultiplicity rages from about 11.4 to about 26.25 and shoot lengthranges from less than 1 cm to about 5 cm.

In some preferred embodiments, the shoot proliferation medium comprisesmodified MS basal culture medium, about 1 mg/L IAA, and about 3%sucrose. This medium is capable of inducing or supporting shootproliferation with a shoot multiplicity of about 20.3 and shoot lengthranging from about 2.5 cm to about 3.0 cm.

In some embodiments, the shoot proliferation medium comprises modifiedMS basal culture medium, about 1 mg/L IAA, and about 1% sucrose. Thismedium is capable of inducing or supporting shoot proliferation with ashoot multiplicity of about 11.4.

In some embodiments, the shoot proliferation medium comprises modifiedMS basal culture medium, about 1 mg/L IAA, and about 2% sucrose. Thismedium is capable of inducing or supporting shoot proliferation with ashoot multiplicity of about 11.4.

In some preferred embodiments, the shoot proliferation medium comprisesmodified MS basal culture medium, about 0.5 mg/L BAP, and about 1.0 mg/LGA₃. This medium is capable of inducing or supporting shootproliferation with a shoot multiplicity of about 26.25 and shoot lengthranging from about 1 cm to about 5 cm.

In some preferred embodiments, the shoot proliferation medium comprisesmodified MS basal culture medium, about 0.5 mg/L BAP, about 1.0 mg/LIAA, and about 3% sucrose. This medium is capable of inducing orsupporting shoot proliferation with a shoot multiplicity of about 21.2and shoot length ranging from about 1 cm to about 5 cm.

In some embodiments of the invention, a third or “rooting” mediumcomprises MS basal culture medium, optionally plant hormones selectedfrom the group consisting of from about 1.0 mg/L to about 5.0 mg/L NAA,from about 1.0 mg/L to about 5.0 mg/L IAA, and from about 1.0 mg/L toabout 5.0 mg/L IBA, and from about 1% to about 3% sucrose. Thesecompositions are capable of inducing or supporting efficient developmentof the root system. Rooting percentages for compositions of theinvention range between 50% to 80% at from about 8 weeks to about 10weeks.

In some preferred embodiments, the rooting medium comprises modified MSbasal culture medium and from about 1.0 mg/L to about 5.0 mg/L IAA. Thismedium is capable of inducing or supporting from about 75% to about 80%rooting at about 8 weeks. These roots are well-developed, white coloredand thick.

In some embodiments, the rooting medium comprises modified MS basalculture medium and from about 1.0 mg/L to about 5.0 mg/L IBA. Thismedium is capable of inducing or supporting from about 60% to about 65%rooting at about 8 weeks. These roots are white colored, thick, andshort.

In some embodiments, the rooting medium comprises modified MS basalculture medium and from about 1.0 mg/L to about 5.0 mg/L NAA. Thismedium is capable of inducing or supporting from about 50% to about 60%rooting at about 10 weeks. These roots are brown colored, sparselydeveloped, and lack hairs.

In one embodiment of the invention, the media of Table 1 is used forestablishment of cultures using shoot apices and axillary bud explants,proliferation of multiple shoots, and rooting regenerated shoots. Theseedlings or plantlets so produced are suitable for transfer to soil.

TABLE 1 Tissue Culture Media Formulation for various stages involved inpropagation of Swertia chirata: Proliferation Establishment of shoots/Rooting of Cultures Development of Shoots Constituents (mg/L) (mg/I)(mg/I) MgSO₄ · 7H₂O 370 370 370 MnSO₄ · H₂O 16.9 16.9 16.9 Zn SO₄ · 7H₂O8.6 8.6 8.6 KNO₃ 1900 1900 1900 NH₄NO₃ 1650 1650 1650 CaCl₂ · 2H₂O 440440 440 KH₂ · PO₄ 170 170 170 H₃BO₃ 6.2 6.2 6.2 KI 0.83 0.83 0.83 CuSO₄· 5H₂O 0.025 0.025 0.025 CoCl₂ · 6H₂O 0.025 0.025 0.025 NaMoO₄ · 2H₂O0.025 0.025 0.025 Glycine 2 2 2 Thiamine HCl 0.1 0.1 0.1 Pyridoxine-HCl0.5 0.5 0.5 Nicotinic acid 0.5 0.5 0.5 PH 5.8 5.8 5.8 Supporting systemAgar (0.8%) i) Agar (0.8%) Agar (0.8%) ii) Liquid Benzyladenine (BAP)0.5-1.0 0.5-1.0 — Gibbrellic acid (GA₃) — 1.0 — Indoleacetic acid (IAA)0.5-1.0 1.0 1.0 Indole butyric acid (IBA) 0.5-1.0 — 1.0 Naphthaleneacetic acid 0.5 — 1.0 (NAA) Sucrose    1-3%    1-3% 1-3%Methods

The invention contemplates the use of standard techniques related to invitro cultivation of plants. See e.g Gelvin S B, Schilperoort R A, VermaD P S, eds., Plant Molecular Biology Manual, 2nd edition, Klewer 1994;Clark M, ed., Plant Molecular Biology, Springer Verlag 1997; Jones P,ed., Plant Molecular Biology, John Wiley & Son Ltd 1997. One of ordinaryskill in the art will appreciate that techniques developed for in vitrocultivation and propagation of lily, Arabidopsis, tobacco, rice andother plants may be useful for practicing the instant invention. Unlessotherwise specified, all in vitro methods were performed usingappropriate aseptic techniques known to those of ordinary skill in theart, such as use of Laminar air flow.

The present invention provides a method for multi-fold increased invitro propagation of Swertia chirata comprising:

-   -   cleaning various explants viz. root, stem, apical and axillary        buds-with-nodes of said plant;    -   sterilizing cleansed explants;    -   treating replicates of 10 explants comprising shoot apices,        axillary buds, stem segments seedlings, and leaf segments;    -   using about 0.8% Agar as support system;    -   testing the efficacy of various explants cultured on first media        culture compositions comprising modified MS basal culture media        and BAP ranging between 0.5 to 1.0 mg/L, and optionally        containing NAA about 0.5 mg/L, IAA ranging between 0.5 to 1.0        mg/L, IBA ranging between 0.5 to 1.0 mg/L, and sucrose ranging        between 1 to 3%, supplemented with various plant growth        regulators, to find an efficient explant source for culture        initiation;    -   measuring the effect of various second media compositions        comprising modified MS basal culture medium, and optionally        containing BAP ranging between 0.5 to 1.0 mg/L, IAA about 1.0        mg/L, GA₃ about 1.0 mg/L, and sucrose concentration ranging        between 1 to 3%, on shoot multiplication and shoot development        after about every 4 weeks;    -   excising the shoot developed in above-mentioned cultures;    -   using excised shoots to find the best or a satisfactory second        media composition for shoot multiplication;    -   incubating the above-mentioned cultures at 20° C. under 8/12 hr        light and dark period;    -   subculturing after about every 4 weeks for the respective media        formulations;    -   excising four weeks long shoot obtained as above;    -   transferring excised shoots into modified MS basal culture        medium with varied concentrations of several auxins ranging        between 1 and 5 mg/L, selected from a group comprising IAA, IBA,        and NAA;    -   using excised roots to find the best or a satisfactory third        media composition comprising modified MS basal culture medium,        optionally containing plant hormones selected from a group        consisting of IAA ranging between 1.0 to 5.0 mg/L, IBA ranging        between 1.0 to 5.0 mg/L, NAA ranging between 1.0 to 5.0 mg/L,        and sucrose concentration ranging between 1 to 3%, for well        developed rooting system; and    -   transferring the above-mentioned in vitro grown plant species        Swertia chirata into varied potting mixtures, showing about 70%        survival rate with vermiculite and sterilized garden soil in        ratio 1:1.

Explants are preferably transferred using standard plant tissue cultureaseptic techniques. Aseptic techniques are typically unnecessary fortransferring plantlets to soil.

Initiation

Naturally growing live Swertia chirata plant material from differentlocations in the Darjeeling hills of India may be collected andidentified. Various explants, e.g. root, stem, apical and axillarybuds-with-nodes from collected plants may be surface sterilized beforeinitiating primary in vitro cultures. The efficacy of various explantscultured on different media combinations and supplemented with variousplant growth regulators may be tested to find out efficient explantsource for culture initiation. Each treatment may consist of replicates,e.g. 10, of different explants (shoot apices, axillary buds, stemsegments seedlings, leaf segments). Axillary buds and shoot apicescultured on media described in Table 1 are the most regenerative andresulted shoot proliferation (FIG. 1). A combination of modified MS+BAP(0.5 modified MS)+NAA (0.5 mg/L) and modified MS+BAP(1 mg/L)+IAA (1mg/L) are most responsive treatments (FIG. 1).

Shoot Proliferation

The effect of various media combinations on shoot multiplication andshoot development (after 4 weeks) may be tested. The excised shootsdeveloped in primary cultures may be cultured on media to test theeffect of a range of concentration of various plant growth regulators,adenine sulphate, gibberellic acid, supplemented to modified MS basalmedium to find the best or a satisfactory treatment for shootmultiplication. The cultures may be incubated at 20° C. under 8/12 hrlight and dark period. Subcultures may be done after every 4 weeks onthe same media formulation.

Table 2 shows the influence of various treatment on shoot number andlength.

TABLE 2 Effect of various media combinations on shoot multiplication andshoot development (After 4 weeks) Media Shoot number Shoot BM* + mg/LShoots/Culture length (cm) modified MS + 0.5 BAP  3.6 <1 modified MS +1.0 IAA + 1% sucrose 11.4 <1 modified MS + 1.0 IAA + 2% sucrose 11.8 <1modified MS + 1.0 IAA + 3% sucrose 20.3 2.5-3.0 ½ modified MS + 1.0IAA + 21.2 <1 0.5 mg BAP + 3% sucrose ½ modified MS + 1.0 IAA +  6.6 <10.5 mg BAP + 3% sucrose modified MS + 1.0 IAA + 5 mg  1.2 <1 glutamine +3% sucrose modified MS + 1.0 IAA + 0.5 mg BAP +  5.4 <1 3% sucrosemodified MS + 1.0 IAA + Ads 10 mg  4.2 <1 modified MS + 1.0 IAA + Ads 20mg  6.8 <1 modified MS + 0.5 mg BAP + GA₃ 1 mg  26.25 1-5

Modified MS basal medium+(1.0 mg/L IAA+3% sucrose) and modified MS basalmedium+[0.5 mg BAP+GA₃(1 mg/L)] results in maximum shoot proliferationand shoot development.

Rooting

The effect of various auxins on rooting percent and duration may bestudied using four-week shoots obtained in the preceding section.

The excised shoot may be transferred to modified MS basal culture mediumwith varied concentrations of several auxins selected from a groupcomprising IAA, IBA, and NAA as shown in Table 3.

TABLE 3 Rooting percent and duration as influenced by different auxinsAuxins^(BM+mg.1−1) Rooting % Rooting (wks) IAA* 1 80 8 2 75 8 5 75 8IBA** 1 60 8 2 62 8 5 60 8 NAA*** 1 50 10 2 50 10 5 60 10 Rootmorphology *White-thick, well developed root system **Whitish thin withshort roots ***Brownish sparsely developed roots without root hairs.

Media containing IAA (1 mg/L) fosters maximum rooting percentage andresults in white colored, thick and well developed root system.

Transfer to Soil

In some embodiments of the invention, in vitro cultivated plants may betransferred to soil. The survival rate of Swertia chirata is about 70%in soil comprising a 1:1 mixture of vermiculite and sterilized gardensoil. The survival rate is about 50% in soil comprising eithervermiculite alone or a 1:1 mixture of soilrite and sterilized gardensoil.

The effect of different potting mixtures on in vitro raised plantletsmay be determined by transferring the plantlets obtained in thepreceding section to soil. Examples of various soils are provided inTable 4.

TABLE 4 Effect of different Potting Mixture on in vitro raised plantletsPotting Mixture Comprising: Survival % Soilrite + Garden soil (1:1) 20Vermiculite + Garden soil (1:1) 20 Peatmoss + Garden soil (1:1)  0Garden soil  8 Garden soil (Sterilized) 20 Sand 40 Soilrite + Perlite(10:1) 30 Vermiculite 50 Peatmoss  0 Soilrite + Garden soil (1:1) 50Sterilized Vermiculite + Garden soil (1:1) 70 Sterilized Peatmoss +Garden soil (1:1) 40 Garden soil is the mixture of soil found in theforest area.

The best percentage of plantlet survival is observed in polyhouse wherevermiculite and garden soil are used in the ratio of 1:1.

EXAMPLES

The present invention is illustrated, but not limited, by the followingexamples. Other examples and embodiments will be apparent to thoseskilled in the art and do not depart from the spirit and scope of theinvention.

Example 1

The experiment consisted of 10 treatments in the form of variouscombination of benzyladenine (BA 0.5-mg/c), IAA, IBA, and α-NAAsupplemented to modified MS based culture medium with 0.8% agar byweight.

Each treatment consisted of 10 replicates of different explants (shootapices, axillary buds, stem segments seedlings, leaf segments).

Axillary buds and shoot apices cultured on media described in Table Iwere most regenerative and resulted shoot proliferation (FIG. 1).

Combination of modified MS+BAP (0.5 ms)+NAA (0.5 mg/L) and modifiedMS+BAP (1 mg/L)+IAA(1 mg/L) were most responsive treatments (FIG. 1).

Example 2

In this experiment the shoot developed in primary cultures were excisedand cultured on media to test the effect of a range of concentration ofvarious plant growth regulators, adenine sulphate, gibberellic acid,supplemented to modified MS basal medium to find the best or asatisfactory treatment for shoot multiplication.

The cultures were incubated at 20° C. under 8/12 hr light and darkperiod. Subculture was done after every 4 weeks on the same mediaformulation.

The results in Table 2 show the influence of various treatment on shootnumber and length.

Among the tested combinations modified MS basal medium+(1.0 mg/L IAA+3%sucrose and 0.5 mg BAP+GA3 (1 mg/L) resulted maximum shoot proliferationand better shoot development.

Example 3

In this experiment there are 3 treatments of auxin IAA, IBA and NAAusing various levels (1-5 mg/L) supplemented to modified MS media,Supporting system Agar (0.8%).

Four weeks long shoot achieved in experiment 2 were excised andtransferred to above referred medium

The results in Table 3 show the influence of various treatment onrooting percentage and duration as influenced by respective treatmentand concentration.

Media containing IAA (1 mg/L) fostered maximum rooting percentage andresulted white thick were root system.

Example 4

Rooted plantlets of Example 3 were transferred to different pottingmixtures. Better percentage of plantlets survived in polyhouse wherevermiculite and garden soil (1:1 ratio) were used (Table 4).

The references cited throughout this application are incorporated hereinin their entirety by reference.

1. A method for cultivating Swertia in vitro comprising: contacting aSwertia explant with a first medium comprising a MS basal culture mediumI, from about 0.5 mg/L to about 1.0 mg/L benzyladenine, from about 1% toabout 3% by weight of sucrose, and at least one auxin selected from thegroup consisting of about 0.5 mg/L naphthalene acetic acid, from about0.5 mg/L to about 1.0 mg/L indole acetic acid, and from about 0.5 mg/Lto about 1.0 mg/L indole butyric acid to produce a primary explant;contacting the primary explant with a second medium comprising a MSbasal culture medium I, about 1.0 mg/L indole acetic acid, about 1.0mg/L gibberellic acid, and from about 1% to about 3% sucrose to producea secondary explant; and contacting the secondary explant with a thirdmedium comprising a MS basal culture medium I, from about 1% to about 3%sucrose, and at least one auxin selected from the group consisting offrom about 1.0 mg/L to about 5.0 mg/L naphthalene acetic acid, fromabout 1.0 mg/L to about 5.0 mg/L indole acetic acid, and from about 1.0mg/L to about 5.0 mg/L indole butyric acid to produce a tertiaryexplant.
 2. The method of claim 1, wherein the Swertia is Swertiachirata.
 3. The method of claim 2, wherein the Swertia chirata isSwertia chirata (Ham).
 4. The method of claim 1, wherein the Swertiaexplant comprises meristematic tissue.
 5. The method of claim 4, whereinthe meristematic tissue is an axillary bud or a shoot apex.
 6. Themethod of claim 1, wherein the first medium further comprises about 0.5mg/L naphthalene acetic acid.
 7. The method of claim 1, wherein thesecond medium further comprises from about 0.5 mg/L to about 1.0 mg/Lbenzyladenine.
 8. The method of claim 1, wherein the first, second,and/or third media further comprise agar.
 9. The method of claim 1,wherein the MS basal culture medium I of the first, second, and/or thirdmedium has a pH of about 5.8.
 10. The method of claim 1 furthercomprising contacting the tertiary explant with soil.
 11. The method ofclaim 1, wherein the primary explant is contacted with the second mediumat about 20° C.
 12. The method of claim 1, wherein the primary explantis contacted with the second medium under at least one cycle of about 8hr light/about 12 hr dark.
 13. The method of claim 1, wherein theduration of contact with the first medium is about 4 weeks.
 14. Themethod of claim 1, wherein the duration of contact with the secondmedium is about 4 weeks.
 15. The method of claim 1, wherein the durationof contact with the third medium is from about 8 weeks to about 10weeks.
 16. The method of claim 1, wherein the duration of contact withthe first medium is about 4 weeks, the duration of contact with thesecond medium is about 4 weeks, and the duration of contact with thethird medium is from about 8 weeks to about 10 weeks.
 17. The method ofclaim 1, wherein plants having a shoot length of from about 1 cm toabout 5 cm and capable of a 70% survival rate on soil are produced inabout 18 weeks or less.
 18. The method of claim 1, wherein the firstmedium comprises MS basal culture medium I, from about 0.5 mg/L to about1.0 mg/L benzyladenine, from about 0.5 mg/L to about 1.0 mg/L indoleacetic acid, from about 0.5 mg/L to about 1.0 mg/L indole butyric acid,about 0.5 mg/L naphthalene acetic acid, and from about 1% to about 3%sucrose; the second medium comprises MS basal culture medium I, fromabout 0.5 mg/L to about 1.0 mg/L benzyladenine, about 1.0 mg/L indoleacetic acid, about 1.0 mg/L gibberellic acid, and from about 1% to about3% sucrose; and the third medium comprises MS basal culture medium I,about 1.0 mg/L indole acetic acid, about 1.0 mg/L indole butyric acid,about 1.0 mg/L naphthalene acetic acid, and from about 1% to about 3%sucrose.
 19. A method for cultivating Swertia chirata in vitrocomprising: contacting primary explants of Swertia chirata with a shootproliferation medium comprising an MS basal culture medium I, from about0.5 mg/L and about 1.0 mg/L benzyladenine, about 1.0 mg/L indole aceticacid, about 1.0 mg/L gibberellic acid, and from about 1% to about 3%sucrose, wherein a multiplicity of from about 11.4 to about 26.25secondary explants of Swertia chirata with a shoot length ranging fromabout 1 cm to about 5 cm is obtained.
 20. A method for rooting Swertiachirata secondary explants in vitro comprising: contacting secondaryexplants of Swertia chirata with a rooting medium comprising an MS basalculture medium I, from about 1% to about 3% sucrose, and at least oneauxin selected from the group consisting of from about 1.0 mg/L to about5.0 mg/L naphthalene acetic acid, from about 1.0 mg/L to about 5.0 mg/Lindole acetic acid, and from about 1.0 mg/L to about 5.0 mg/L indolebutyric acid; wherein from about 50% to about 80% of the secondaryexplants of Swertia chirata produce white roots in less than about 10weeks.
 21. A method for cultivating Swertia in vitro comprising:contacting a Swertia chirata shoot apex or axillary bud explant with afirst medium comprising MS basal culture medium I, from about 0.5 mg/Lto about 1.0 mg/L benzyladenine, from about 0.5 mg/L to about 1.0 mg/Lindole acetic acid, from about 0.5 mg/L to about 1.0 mg/L indole butyricacid, about 0.5 mg/L naphthalene acetic acid, and from about 1% to about3% sucrose to produce a primary explant; contacting this primary explantwith a second medium comprising MS basal culture medium I, from about0.5 mg/L to about 1.0 mg/L benzyladenine, about 1.0 mg/L indole aceticacid, about 1.0 mg/L gibberellic acid, and from about 1% to about 3%sucrose to produce a secondary explant; and contacting this secondaryexplant with a third medium comprising MS basal culture medium I, about1.0 mg/L indole acetic acid, about 1.0 mg/L indole butyric acid, about1.0 mg/L naphthalene acetic acid, and from about 1% to about 3% sucroseto produce a tertiary explant; wherein contacting the primary explantwith the second medium occurs at about 20° C. and under at least onecycle of about 8 hr light/about 12 hr dark.
 22. A method of producing aSwertia chirata plant with a shoot length of from about 1 cm to 5 cm invitro from shoot apex or axillary bud explants in about 18 weeks or lesscomprising: contacting a Swertia chirata shoot apex or axillary budexplant with a first medium comprising MS basal culture medium I, fromabout 0.5 mg/L to about 1.0 mg/L benzyladenine, from about 0.5 mg/L toabout 1.0 mg/L indole acetic acid, from about 0.5 mg/L to about 1.0 mg/Lindole butyric acid, about 0.5 mg/L naphthalene acetic acid, and fromabout 1% to about 3% sucrose to produce a primary explant; contactingthis primary explant with a second medium comprising MS basal culturemedium I, from about 0.5 mg/L to about 1.0 mg/L benzyladenine, about 1.0mg/L indole acetic acid, about 1.0 mg/L gibberellic acid, and from about1% to about 3% sucrose to produce a secondary explant; and contactingthis secondary explant with a third medium comprising MS basal culturemedium I, about 1.0 mg/L indole acetic acid, about 1.0 mg/L indolebutyric acid, about 1.0 mg/L naphthalene acetic acid, and from about 1%to about 3% sucrose to produce a tertiary explant; wherein contactingthe primary explant with the second medium occurs at about 20° C. andunder at least one cycle of about 8 hr light/about 12 hr dark.